The present application provides multiplex drop-off digital polymerase chain reaction (dPCR) assays, methods, systems, and kits. The methods described herein are useful in a variety of applications, such as detection of microsatellite instability and quantification of site-specific genome-edited products.
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1. A method for quantification of wildtype and/or mutant sequences at R number of target regions in a sample comprising nucleic acid molecules using R number of detection channels and R number of probe pairs corresponding to the R number of target regions, wherein each of the R number of probe pairs comprises a reference probe having a reference label and a drop-off probe having a drop-off label, wherein the set of reference labels and the set of drop-off labels corresponding to the R number of probe pairs are circular permutations with respect to each other, wherein the nucleic acid molecules are distributed among a plurality of partitions of the sample, and wherein substantially all partitions each comprises the R number of probe pairs, wherein a first probe pair of the R number of probe pairs comprises: a first reference probe comprising a first reference sequence (r 1 ) and a first reference label detectable via a first detection channel (X 1 ), and a first drop-off probe comprising a first drop-off sequence (w 1 ) and a first drop-off label detectable via a second detection channel (X 2 ); wherein a second probe pair of the R number of probe pairs comprises: a second reference probe comprising a second reference sequence (r 2 ) and a second reference label detectable via the second detection channel (X 2 ), and a second drop-off probe comprising a second drop-off sequence (w 2 ) and a second drop-off label detectable via a third detection channel (X 3 ); wherein, when R is strictly larger than 3, an i-th probe pair (2≤i≤R) of the R number of probe pairs comprises: an i-th reference probe comprising an i-th reference sequence (r i ) and an i-th reference label detectable via an i-th detection channel (X i ), and an i-th drop-off probe comprising an i-th drop-off sequence (w i ) and an i-th drop-off label detectable via an (i+l)-th detection channel (X i+1 ); wherein, when R is strictly larger than 2, a R-th probe pair of the R number of probe pairs comprises: a R-th reference probe comprising a R-th reference sequence (r R ) and a R-th reference label associated with a R-th detection channel (X R ), and a R-th drop-off probe comprising a R-th drop-off sequence (w R ) and a R-th drop-off label detectable by the first detection channel (X 1 ); wherein the drop-off sequence of each probe pair is complementary to a wildtype sequence at a target region corresponding to the respective probe pair; wherein the reference sequence of each probe pair is complementary to a wildtype sequence at an adjacent reference region upstream or downstream to the target region corresponding to the respective probe pair; wherein the detection channels X 1 -X R are different from each other; wherein the method comprises: detecting hybridization of reference probes of the R number of probe pairs to nucleic acid molecules comprising wildtype sequences at the reference regions or amplicons thereof in the plurality of partitions via each of the detection channels X 1 -X R ; and detecting hybridization of drop-off probes of the R number of probe pairs to nucleic acid molecules comprising wildtype sequences at the target regions or amplicons thereof in the plurality of partitions via each of the detection channels X 1 -X R ; thereby providing quantification of wildtype and/or mutants sequences at the R number of target regions in the sample.
2. The method of claim 1 , further comprising: obtaining a first count of one or more partitions that each produces a positive signal via the i-th detection channel and negative signals via any other of the detection channels X 1 -X R ; obtaining a second count of one or more partitions that each produces negative signals via all of the detection channels X 1 -X R ; and calculating a mutant probability ({circumflex over (P)}(m i )) that a given partition contains a mutant sequence at the target region corresponding to the i-th probe pair, wherein the mutant probability is based on a ratio between the first count and a sum of the first count and the second count.
3. The method of claim 2 , further comprising determining: (i) an estimated concentration of the mutant sequences at the target region corresponding to the i-th probe pair in the sample based on the mutant probability; and (ii) a confidence interval and/or an uncertainty measure associated with the estimated concentration of the mutant sequences at the target region corresponding to the i-th probe pair in the sample.
4. The method of claim 2 , further comprising calculating a wildtype probability that a given partition contains a wildtype sequence at the target region corresponding to the i-th probe pair, wherein the wildtype probability is based on the mutant probability corresponding to the i-th probe pair and the mutant probability corresponding to the (i+1)-th probe pair, wherein the (i+1)-th probe pair refers to the first probe pair if i=R.
5. The method of claim 4 , further comprising determining: (i) an estimated concentration of the wildtype sequence at the target region corresponding to the i-th probe pair in the sample based on the wildtype probability; and (ii) a confidence interval and/or a uncertainty measure associated with the estimated concentration of the wildtype sequence at the target region corresponding to the i-th probe pair in the sample.
6. The method of claim 1 , wherein R is between 2 and 6.
7. The method of claim 6 , wherein R is 3.
8. The method of claim 1 , further comprising adjusting the concentration of nucleic acid molecules in the sample based on a count of partitions that each produces a positive signal via three or more of the detection channels X 1 -X R , wherein: (i) if the count is larger than a pre-determined value, the adjusting is decreasing the concentration of the nucleic acid molecules in the sample by diluting the sample; or (ii) if the count is smaller than a pre-determined value, the adjusting is increasing the concentration of the nucleic acid molecules in the sample by concentrating the sample.
9. The method of claim 1 , wherein substantially all partitions each further comprises: an allele-specific (AS) probe comprising an AS label and an oligonucleotide AS sequence complementary to an allelic sequence at a target region, wherein the AS label is detectable via a detection channel that is different from the detection channels corresponding to the reference probes and the drop-off probes of the R number of probe pairs; and wherein the method further comprises: detecting hybridization of the AS probe to nucleic acid molecules comprising the allelic sequence at the target region or amplicons thereof in the plurality of partitions, thereby providing quantification of the allelic sequence at the target region in the sample.
10. The method of claim 1 , wherein each of the reference probes and the drop-off probes has a single detectable label.
11. The method of claim 1 , wherein the reference labels and drop-off labels are fluorophores.
12. The method of claim 11 , wherein one or more different detection channels have different excitation wavelength ranges and/or different emission wavelength ranges.
13. The method of claim 11 , wherein one or more different detection channels share the same excitation and/or emission wavelength ranges, but are associated with different fluorescence intensities.
14. The method of claim 11 , wherein R is 3, and wherein the first reference label, the second reference label and the third reference label are selected from the group consisting of Cy3, FAM and Cy5, or wherein the first reference label, the second reference label and the third reference label are selected from the group consisting of FAM, HEX and Cy5.
15. The method of claim 1 , wherein the target regions are mutation hotspot regions in one or more genes selected from the group consisting of EGFR, NRAS, KRAS, ESR1, and BRAF.
16. The method of claim 1 , wherein each partition further comprises: (a) R number of primer sets corresponding to the R number of target regions, and (b) a DNA-dependent DNA polymerase; wherein each primer set of the R number of primer sets comprises a forward oligonucleotide primer and a reverse oligonucleotide primer suitable for amplifying a target fragment comprising a target region corresponding to the primer set and the reference region corresponding to the target region; wherein the method comprises amplifying the target fragments from the nucleic acid molecules in the plurality of partitions; and wherein the detecting comprises detecting hybridization of the reference probes and the drop-off probes to amplicons of the target fragments.
17. The method of claim 1 , wherein the nucleic acid molecules are genomic DNA molecules, tumor DNA molecules, or cDNA molecules.
18. The method of claim 1 , wherein the R number of target regions are microsatellite sequence loci.
19. The method of claim 1 , wherein the nucleic acid molecules are genomic DNA in a sample of cells, wherein the cells have been contacted with a site-specific genome-editing reagent configured to cleave target sites in the R number of target regions, and wherein the mutant sequences are non-homologous end joining (NHEJ) edited sequences at the R number of target regions.
20. The method of claim 19 , wherein the site-specific genome-editing reagent comprises a Cas nuclease, a Transcription activator-like effector nuclease (TALEN), or a Zinc-finger nuclease.
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September 4, 2020
July 13, 2021
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